Neuroblastoma is a childhood cancer that is derived from precursor cells of the adrenosympathetic system, originating in the adrenal medulla or sympathetic ganglia. To gain insight into the mechanism of action of the MYCN gene in neuroblastoma pathogenesis, we used RNA interference (RNAi) to suppress MYCN expression. The MYCN gene was transiently suppressed using 27-mer siLentMer™*

Human IMR-32 neuroblastoma cells were transfected with two anti-MYCN siLentMer siRNA duplexes and the level of MYCN transcript was determined 48 h posttransfection by rt-qPCR using five different primer pairs (Figure 1A). Results summarized in Figure 1 show variable levels of MYCN silencing observed with the different primer pairs (Figure 1B). These results indicate the importance of primer location for evaluation of siRNA silencing efficiency, consistent with a previously published study (2). The target mRNA sequence is cleaved by the RNA-induced silencing complex near the center of the region complementary to the guiding siRNA (3) and complete nucleolytic degradation of the resulting fragments does not always occur. Therefore, using primers that do not span the siRNA target sequence may result in an underestimation of siRNA silencing efficiency.

rt-qPCR is the method of choice for accurate, sensitive, and specific quantitation of nucleic acid sequences. It provides a convenient and reliable tool for evaluating knockdown efficiency and functional effects of RNAi-mediated gene silencing, as demonstrated by the use of this tool for monitoring knockdown of MYCN, a key gene in neuroblastoma pathogenesis. Successful application of rt-qPCR requires careful attention to all of the steps in the assay workflow, including primer design and evaluation, template preparation, normalization strategy, and data analysis. Correspondence

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